Methods of Treatment Using G-CSF Protein Complex

ABSTRACT

This disclosure provides a method of preventing, alleviating, or treating a condition (i.e., neutropenia) in a patient in need thereof, the condition characterized by compromised white blood cell production in the patient. The method includes administering to the patient a therapeutically effective amount of a protein complex comprising a modified human granulocyte-colony stimulating factor (hG-CSF) covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer. The non-peptidyl polymer is site-specifically linked to an N-terminus of the immunoglobulin Fc region, and the modified hG-CSF comprises substitutions in at least one of Cys17 and Pro65.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/428,351 filed May 31, 2019, the disclosure of which ishereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to protein complexes, pharmaceuticalcompositions, and methods of use thereof for treating, preventing, orreducing the risk of developing a condition, such as neutropenia. Theprotein complex can be formed by linking an immunoglobulin Fc region toa physiologically active polypeptide via a non-peptidyl polymer, inwhich the non-peptidyl polymer is linked to the immunoglobulin Fcregion.

BACKGROUND OF THE INVENTION

Neutropenia is a relatively common disorder most often associated withchemotherapy treatments, adverse drug reactions, or autoimmunedisorders. Chemotherapy-induced neutropenia is common toxicity caused bythe administration of anticancer drugs. It is associated withlife-threatening infections and may alter the chemotherapy schedule,thus impacting on early and long-term clinical outcome. FebrileNeutropenia is major dose-limiting toxicity of myelosuppressivechemotherapy regimens such as docetaxel, doxorubicin, cyclophosphamide(TAC); dose-dense doxorubicin plus cyclophosphamide (AC), with orwithout subsequent weekly or semiweekly paclitaxel; and docetaxel pluscyclophosphamide (TC). It usually leads to prolonged hospitalization,intravenous administration of broad-spectrum antibiotics, and is oftenassociated with significant morbidity and mortality. About 25% to 40% oftreatment naïve patients develop febrile neutropenia with commonchemotherapy regimens in the absence of G-CSF support.

Current therapeutic modalities employ granulocyte colony-stimulatingfactor (G-CSF) and/or antibiotic agents to combat this condition.However, G-CSF or its other polypeptide derivatives are easy to denatureor easily de-composed by proteolytic enzymes in blood to be readilyremoved through the kidney or liver. Therefore, to maintain the bloodconcentration and titer of the G-CSF containing drugs, it is necessaryto frequently administer the protein drug to patients, which causesexcessive suffering in patients. To solve such problems, G-CSF waschemically attached to polymers having a high solubility such aspolyethylene glycol (“PEG”), thereby increasing its blood stability andmaintaining suitable blood concentration for a longer time.

However, binding of PEG to G-CSF, even though may increase bloodstability, does dramatically reduce the titer needed for optimalphysiologic effect. Thus there is a need to address this shortcoming inthe art.

Thus, there is a strong need for new formulations and methods of usewhere the new G-CSF containing protein complex can stay stable anddramatically improve patient outcome.

SUMMARY OF THE INVENTION

The present invention is directed to methods of using a G-CSF containinga more stable protein complex that can be easily prepared andadministered to patients at risk of developing neutropenia, and maintaina serum concentration that achieves the optimal therapeutic outcome.Another aspect of the present invention is directed to a protein complexprepared by linking a physiologically active polypeptide and animmunoglobulin Fc fragment via a non-peptidyl polymer, in which thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc fragment.

In one aspect, this disclosure provides a method of preventing,alleviating or treating a condition in a patient in need thereof. Themethod comprises administering to the patient a therapeuticallyeffective amount of a protein complex comprising a modified humangranulocyte-colony stimulating factor (hG-CSF) covalently linked to animmunoglobulin Fc region via a non-peptidyl polymer, wherein thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc region and the modified hG-CSF comprises substitutionsin at least one of Cys17 and Pro65.

The condition is characterized by compromised white blood cellproduction in the patient. For example, the condition can be one of:reduced hematopoietic function, reduced immune function, reducedneutrophil count, reduced neutrophil mobilization, mobilization ofperipheral blood progenitor cells, sepsis, severe chronic neutropenia,febrile neutropenia, bone marrow transplants, infectious diseases,leucopenia, thrombocytopenia, anemia, enhancing engraftment of bonemarrow during transplantation, enhancing bone marrow recovery intreatment of radiation, chemical or chemotherapeutic induced bone marrowaplasia or myelosuppression, and acquired immune deficiency syndrome.

In some embodiments, the condition can be severe chronic neutropenia. Insome embodiments, the condition can be febrile neutropenia. In someembodiments, compromised white blood cell production can be a result ofchemotherapy, radiation therapy, or idiopathic thrombocytopenia purpura.

In some embodiments, the protein complex can be administered after thepatient is treated with adjuvant or neoadjuvant chemotherapy. In someembodiments, the protein complex can be administered between 1 and 5days after the patient is treated with adjuvant or neoadjuvantchemotherapy. In some embodiments, the adjuvant or neoadjuvantchemotherapy can be a combination of docetaxel and cyclophosphamide.

In some embodiments, a second dose of the protein complex can beadministered between 15 and 25 days after a first dose of the proteincomplex is administered to the patient.

In some embodiments, the therapeutically effective amount is a unitdosage form selected from: 25 μg/kg, 50 μg/kg, 100 μg/kg, and 200 μg/kg.In some embodiments, the therapeutically effective amount is 13.2 mg ofthe protein complex in a 0.6 mL dosage volume.

In some embodiments, the method further comprises administering to thepatient a therapeutically effective amount of a second agent (e.g., ananti-cancer agent).

In some embodiments, the substitution at Cys17 can be Cys17Ser, and thesubstitution at Pro65 can be Pro65Ser.

In some embodiments, the modified human G-CSF comprises a polypeptidesequence of SEQ ID NOs: 1. In some embodiments, the immunoglobulin Fcregion comprises a polypeptide sequence of SEQ ID NO: 2.

In some embodiments, both ends of the non-peptidyl polymer arerespectively linked to the modified human G-CSF and the immunoglobulinFc region through reactive groups by a covalent bond.

In some embodiments, the immunoglobulin Fc region can be characterizedby: (a) the immunoglobulin Fc region is aglycosylated; (b) theimmunoglobulin Fc region consists of CH2 and CH3 domains; (c) theimmunoglobulin Fc region comprises one of CH2, CH3, and CH4 domains; (d)the immunoglobulin Fc region further comprises a hinge region; or (e)the immunoglobulin Fc region is an immunoglobulin Fc fragment derivedfrom IgG, IgA, IgD, IgE, or IgM.

In some embodiments, the immunoglobulin Fc region can be furthercharacterized by: (a) each domain of the immunoglobulin Fc fragment is ahybrid of domains, in which each domain has a different origin derivedfrom immunoglobulins selected from the group consisting of IgG, IgA,IgD, IgE, and IgM; (b) the immunoglobulin Fc fragment is a dimer ormultimer consisting of single chain immunoglobulins comprising domainshaving the same origin; (c) the immunoglobulin Fc fragment is an IgG4 Fcfragment; or (d) the immunoglobulin Fc fragment is a human aglycosylatedIgG4 Fc fragment.

In some embodiments, the non-peptidyl polymer is: (a) selected from thegroup consisting of polyethylene glycol, polypropylene glycol, anethylene glycol-propylene glycol copolymer, polyoxyethylated polyol,polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, abiodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and acombination thereof; or (b) polyethylene glycol. In some embodiments,the polyethylene glycol has a molecular weight of 3.4 kDa.

In some embodiments, the reactive group of the non-peptidyl polymer isselected from the group consisting of an aldehyde group, a maleimidegroup, and a succinimide derivative. For example, the aldehyde group canbe a propionaldehyde group or a butyraldehyde group. The succinimidederivative can be succinimidyl carboxymethyl, succinimidyl valerate,succinimidyl methylbutanoate, succinimidyl methylpropionate,succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide,or succinimidyl carbonate.

In some embodiments, the non-peptidyl polymer has an aldehyde group as areactive group at both ends. In some embodiments, the non-peptidylpolymer has an aldehyde group and a maleimide group as a reactive groupat both ends, respectively. In some embodiments, the non-peptidylpolymer has an aldehyde group and a succinimide group as a reactivegroup at both ends, respectively.

In some embodiments, each end of the non-peptidyl polymer is linked tothe N-terminus of the immunoglobulin Fc region and an N-terminus, aC-terminus, or a free reactive group of a lysine residue, a histidineresidue, or a cysteine residue of the modified human G-CSF,respectively.

In another aspect, this disclosure provides a method for treating orpreventing neutropenia in a patient receiving chemotherapy. The methodcomprises comprising administering to said patient a protein complexcomprising a physiologically active polypeptide linked to animmunoglobulin Fc region via a non-peptidyl polymer, wherein thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc region. In some embodiments, the physiologicallyactive polypeptide is G-CSF.

In some embodiments, both ends of the non-peptidyl polymer isrespectively linked to the physiologically active polypeptide and theimmunoglobulin Fc region through reactive groups by a covalent bond.

In some embodiments, the immunoglobulin Fc region is an immunoglobulin.In some embodiments, the immunoglobulin Fc region is aglycosylated. Insome embodiments, the immunoglobulin Fc region comprises any one of CH2,CH3, and CH4 domains. In some embodiments, the immunoglobulin Fc regionconsists of CH2 and CH3 domains. The immunoglobulin Fc region canfurther comprise a hinge region.

In some embodiments, each domain of the immunoglobulin Fc fragment is ahybrid of domains, in which each domain has a different origin derivedfrom immunoglobulins selected from the group consisting of IgG, IgA,IgD, IgE, and IgM.

In some embodiments, the protein complex can be administered to thepatient within about 26 hours, 24 hours, 22 hours, 18 hours, 12 hours, 6hours, about 5 hours, 2 hours, or 1 hour of the completion ofchemotherapy.

In another aspect, the present invention provides a method of preparingthe protein complex in a pharmaceutical composition for improving invivo duration and stability of the physiologically active polypeptide,the composition including the protein complex as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows results of SDS-PAGE and western blotting of a^(17,65)Ser-G-CSF-PEG-Fc complex which was prepared by N-terminalreaction of an immunoglobulin Fc region.

FIG. 1B shows a result of peptide mapping for analyzing Fc regionN-terminal binding of a ^(17,65)Ser-G-CSF-PEG-Fc complex which wasprepared by N-terminal reaction of an immunoglobulin Fc region.

FIG. 2 shows that the reduction in the incidence of severe neutropeniain the ^(17,65)Ser-G-CSF-PEG-Fc (EFLAPEGRASTIM) arm that isstatistically significant.

FIG. 3 shows that neutropenic complications, including hospitalizationsdue to severe neutropenia and/or use of anti-infective for neutropenia,are significantly less in the EFLAPEGRASTIM arm.

DETAILED DESCRIPTION OF THE INVENTION

Broadly speaking, the present disclosure provides methods of preventing,alleviating, prophylactically treating, and treating a patient having acondition characterized by the compromised white blood cell production.The method includes administering to the patient in need of suchtreatment a therapeutically effective amount of a protein complexcomprising a physiologically active polypeptide, such as a modifiedhuman granulocyte-colony stimulating factor (hG-CSF), covalently linkedto an immunoglobulin Fc region via a non-peptidyl polymer. Thenon-peptidyl polymer can be site-specifically linked to an N-terminus ofthe immunoglobulin Fc region, and the modified hG-CSF includessubstitutions in at least one of Cys17 and Pro65.

In another aspect, the present disclosure provides a method forincreasing the number of granulocytes in eligible patients for a bonemarrow transplant. The method includes administering to the patient inneed of such treatment a therapeutically effective amount of a proteincomplex comprising a modified human granulocyte-colony stimulatingfactor (hG-CSF) covalently linked to an immunoglobulin Fc region via anon-peptidyl polymer, wherein the non-peptidyl polymer issite-specifically linked to an N-terminus of the immunoglobulin Fcregion and the modified hG-CSF comprises substitutions in at least oneof Cys17 and Pro65.

In yet another aspect, the present disclosure provides a method forincreasing stem cell production in a patient. The method includesadministering to the patient in need of such treatment a therapeuticallyeffective amount of a protein complex comprising a modified humangranulocyte-colony stimulating factor (hG-CSF) covalently linked to animmunoglobulin Fc region via a non-peptidyl polymer, wherein thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc region and the modified hG-CSF comprises substitutionsin at least one of Cys17 and Pro65.

In yet another aspect, the present disclosure provides a method forincreasing the number of hematopoietic progenitor cells in a patient inneed that include those undergoing chemotherapy or those who are donorsof stem cells to other patients. The method includes administering tothe patient a therapeutically effective amount of a protein conjugatecomprising a modified human granulocyte-colony stimulating factor(hG-CSF) covalently linked to an immunoglobulin Fc region via anon-peptidyl polymer, wherein the non-peptidyl polymer issite-specifically linked to an N-terminus of the immunoglobulin Fcregion and the modified hG-CSF comprises substitutions in at least oneof Cys17 and Pro65.

In some embodiments, the conditions to be treated include reducedhematopoietic function, reduced immune function, reduced neutrophilcount, reduced neutrophil mobilization, mobilization of peripheral bloodprogenitor cells, sepsis, severe chronic neutropenia, bone marrowtransplants, infectious diseases, leucopenia, thrombocytopenia, anemia,enhancing engraftment of bone marrow during transplantation, enhancingbone marrow recovery in treatment of radiation, chemical orchemotherapeutic induced bone marrow aplasia or myelosuppression, andacquired immune deficiency syndrome. In one embodiment, the condition isa myelosuppression, neutropenia, or preferably febrile neutropenia.

In another aspect, the present disclosure provides a method forpreventing, alleviating, prophylactically treating, and treatinginfection as manifested by neutropenia (e.g., febrile neutropenia) inthe patient with non-myeloid malignancies receiving myelosuppressiveanticancer drugs. The method includes administering to the patient atherapeutically effective amount of a protein complex comprising amodified human granulocyte-colony stimulating factor (hG-CSF) covalentlylinked to an immunoglobulin Fc region via a non-peptidyl polymer,wherein the non-peptidyl polymer is site-specifically linked to anN-terminus of the immunoglobulin Fc region and the modified hG-CSFcomprises substitutions in at least one of Cys17 and Pro65.

In some embodiments, the compromised white blood cell production is aresult of chemotherapy, radiation therapy, adjuvant or neoadjuvantchemotherapy, or idiopathic thrombocytopenia purpura. In certainembodiments, the adjuvant or neoadjuvant chemotherapy is a combinationof docetaxel and cyclophosphamide. In other embodiments, the therapeuticeffective amount is a unit dosage form selected from: 25 μg/kg, 50μg/kg, 100 μg/kg, and 200 μg/kg. In certain embodiments, the presentmethodology, further includes administering to the patient atherapeutically effective amount of a second agent, such as ananti-cancer agent. In certain embodiments, the modified G-CSF is by wayof a substitution at Cys17 is Cys17Ser. In other embodiments, thesubstation at Pro65 is Pro65Ser.

In some embodiments, the immunoglobulin Fc region comprises apolypeptide sequence of SEQ ID NO: 1. In some embodiments, the modifiedG-CSF comprises a polypeptide sequence of SEQ ID NO: 2.

OTHER SEQ ID NO SEQUENCE INFORMATION SEQ ID NO: 1TPLGPASSLPQSFLLKSLEQVRKIQGDGAALQEK G-CSF (17Ser andLCATYKLCHPEELVLLGHSLGIPWAPLSSCSSQA 65Ser)LQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTL DTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVL RHLAQP SEQ ID NO: 2PSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC ImmunoglobulinVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE Fc region (IgG4)EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In some embodiments, the protein complex employed in the present methodscontain (a) each domain of the immunoglobulin Fc fragment is a hybrid ofdomains, in which each domain has a different origin derived fromimmunoglobulins selected from the group consisting of IgG, IgA, IgD,IgE, and IgM; (b) the immunoglobulin Fc fragment is a dimer or multimerconsisting of single chain immunoglobulins comprising domains having thesame origin; (c) the immunoglobulin Fc fragment is an IgG4 Fc fragment;or (d) the immunoglobulin Fc fragment is a human aglycosylated IgG4 Fcfragment.

In certain embodiments, the non-peptidyl polymer is selected from thegroup consisting of polyethylene glycol, polypropylene glycol, anethylene glycol-propylene glycol copolymer, polyoxyethylated polyol,polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, abiodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and acombination thereof. In a preferred embodiment, the non-peptidyl polymeris polyethylene glycol.

In another aspect, the present disclosure provides a method for treatingor preventing neutropenia in a patient receiving chemotherapy. Themethod comprises administering to said patient a protein complexcomprising a modified G-CSF linked to an immunoglobulin Fc region via anon-peptidyl polymer, wherein the non-peptidyl polymer issite-specifically linked to an N-terminus of the immunoglobulin Fcregion. In some embodiments, both ends of the non-peptidyl polymer arerespectively linked to the physiologically active polypeptide and theimmunoglobulin Fc region through reactive groups by a covalent bond. Ina preferred embodiment, the immunoglobulin Fc region is aglycosylated.

In some embodiments, the G-CSF complex composition is administered tothe patient within about 26 hours, 24 hours, 22 hours, 18 hours, 12hours, 6 hours, 5 hours, 2 hours, 1 hour, or half an hour of thecompletion of chemotherapy.

In some embodiment, the present invention provides the protein complexin which the immunoglobulin Fc region comprises one of CH2, CH3, and CH4domains. For example, the immunoglobulin Fc region can include CH2 andCH3 domains. In some embodiments, the immunoglobulin Fc region furtherincludes a hinge region.

In some embodiments, the immunoglobulin Fc region is an immunoglobulinFc fragment derived from IgG, IgA, IgD, IgE, or IgM. In someembodiments, each domain of the immunoglobulin Fc fragment is a hybridof domains and each domain has a different origin derived fromimmunoglobulins selected from the group consisting of IgG, IgA, IgD,IgE, and IgM. In some embodiments, the immunoglobulin Fc fragment is adimer or multimer consisting of single chain immunoglobulins comprisingdomains having the same origin. In some embodiments, the immunoglobulinFc fragment is an IgG4 Fc fragment.

In some embodiments, the non-peptidyl polymer can be one of polyethyleneglycol, polypropylene glycol, an ethylene glycol-propylene glycolcopolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide,dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipidpolymer, chitin, hyaluronic acid, and a combination thereof, preferablythe non-peptidyl polymer is polyethylene glycol. In some embodiments,the non-peptidyl polymer is 3.4 kDa polyethylene glycol.

In some embodiments, the reactive group of the non-peptidyl polymer canbe one of an aldehyde group, a maleimide group, and a succinimidederivative. The aldehyde group can be a propionaldehyde group or abutyraldehyde group. The succinimide derivative can be succinimidylcarboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate,succinimidyl methylpropionate, succinimidyl butanoate, succinimidylpropionate, N-hydroxysuccinimide, or succinimidyl carbonate.

In some embodiments, the non-peptidyl polymer has an aldehyde group as areactive group at both ends. In some embodiments, the non-peptidylpolymer has an aldehyde group and a maleimide group as a reactive groupat both ends, respectively. In some embodiments, the non-peptidylpolymer has an aldehyde group and a succinimide group as a reactivegroup at both ends, respectively.

In some embodiments, the present invention provides the protein complexin which each end of the non-peptidyl polymer is linked to theN-terminus of the immunoglobulin Fc region; and the N-terminus,C-terminus, or a free reactive group of a lysine residue, a histidineresidue, or a cysteine residue of the physiologically activepolypeptide, respectively.

In another aspect, this disclosure provides a method for treating orpreventing neutropenia in a patient receiving chemotherapy. The methodcomprises comprising administering to said patient a protein complexcomprising a physiologically active polypeptide linked to animmunoglobulin Fc region via a non-peptidyl polymer, wherein thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc region.

In some embodiments, the physiologically active polypeptide can be oneof a hormone, a cytokine, an enzyme, an antibody, a growth factor, atranscription factor, a blood coagulation factor, a vaccine, astructural protein, a ligand protein, and a receptor. In someembodiments, the physiologically active polypeptide is G-CSF.

In another aspect, the present disclosure also provides a method ofpreparing the protein complex. The method comprises:

(a) preparing a protein complex by linking at least one non-peptidylpolymer having a reactive group at both ends, at least onephysiologically active polypeptide, and at least one immunoglobulin Fcregion by a covalent bond, and

(b) isolating the protein complex, essentially including the covalentlylinked physiologically active polypeptide, non-peptidyl polymer, andimmunoglobulin Fc region prepared in step (a), in which the non-peptidylpolymer is linked to the N-terminus of the immunoglobulin Fc fragment.

A specific embodiment of the present invention provides the preparationmethod, in which step (a) comprises:

(a1) preparing a conjugate by linking one end of the non-peptidylpolymer to the immunoglobulin Fc region or the physiologically activepolypeptide by a covalent bond; and

(a2) isolating the conjugate prepared in step (a1) and linking the otherend of the non-peptidyl polymer of the isolated conjugate to the otherof the immunoglobulin Fc region and the physiologically activepolypeptide by a covalent bond.

Another specific embodiment of the present invention provides thepreparation method in which in step (a1), the reaction mole ratiobetween the physiologically active polypeptide and the non-peptidylpolymer is in the range from 1:1 to 1:30, and the reaction mole ratiobetween the immunoglobulin Fc fragment and the non-peptidyl polymer isin the range from 1:1 to 1:20.

Still another specific embodiment of the present invention provides thepreparation method in which step (a1) is performed in a pH conditionfrom 4.0 to 9.0.

Still another specific embodiment of the present invention provides thepreparation method in which step (a1) is performed at a temperature from4.0° C. to 25° C.

Still another specific embodiment of the present invention provides thepreparation method in which in step (a1), the reaction concentration ofthe immunoglobulin Fc region or physiologically active polypeptide is inthe range from 0.1 mg/mL to 100 mg/mL.

Still another specific embodiment of the present invention provides thepreparation method in which in step (a2), the reaction mole ratiobetween the conjugate and the immunoglobulin Fc region or thephysiologically active polypeptide is in the range from 1:0.1 to 1:20.

Still another specific embodiment of the present invention provides thepreparation method in which step (a2) is performed in a pH conditionfrom 4.0 to 9.0.

Still another specific embodiment of the present invention provides thepreparation method in which step (a2) is performed at a temperature from4.0° C. to 25° C.

Still another specific embodiment of the present invention provides thepreparation method in which in step (a2), the concentration of theimmunoglobulin Fc region or physiologically active polypeptide is in therange from 0.1 mg/mL to 100 mg/mL.

Still another specific embodiment of the present invention provides thepreparation method in which step (a1) and step (a2) are performed in thepresence of a reducing agent.

Still another specific embodiment of the present invention provides thepreparation method in which the reducing agent is selected from thegroup consisting of sodium cyanoborohydride (NaCNBH₃), sodiumborohydride, dimethylamine borate, and pyridine borate.

Still another specific embodiment of the present invention provides thepreparation method in which in step (a2), the isolation is performed bya single or combined purification method selected from the groupconsisting of anion exchange chromatography, cation exchangechromatography, hydrophobic chromatography, affinity chromatography, andsize exclusion chromatography.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the anion exchangechromatography resin is any one selected from the group consisting ofquaternary ammonium (Q), quaternary aminoethyl (QAE), dicthylaminocthyl(DEAE), polyethylene amine (PEI), dimethyl-laminomethyl (DMAE), andtrimethylaminoethyl (TMAE).

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the cation exchangechromatography resin is any one selected from the group consisting ofmethylsulfonate (S), sulfopropyl (SP), carboxymethyl (CM), sulfoethyl(SE), and polyaspartic acid.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the hydrophobicchromatography resin is any one selected from the group consisting ofphenyl, octyl, (iso)propyl, butyl, and ethyl.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the affinitychromatography resin is any one selected from the group consisting ofprotein A, heparin, blue, benzamidine, metal ions (cobalt, nickel, andcopper), and an antibody to a part or the entirety of constitutingcomponents of the protein complex, in which both ends of thenon-peptidyl polymer are respectively conjugated to the immunoglobulinFc region and the physiologically active polypeptide.

Still another specific embodiment of the present invention provides thepreparation method in which the resin of the size exclusionchromatography is selected from the group consisting of Superdex,Sephacryl, Superpose, and Sephadex.

Still another specific embodiment of the present invention provides thepreparation method in which the isolating the protein complex of step(b) is performed by a single or combined method selected from the groupconsisting of anion exchange chromatography, cation exchangechromatography, hydrophobic chromatography, affinity chromatography, andsize exclusion chromatography.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the anion exchangechromatography resin is any one selected from the group consisting ofquaternary ammonium (Q), quaternary aminoethyl (QAE), diethylaminoethyl(DEAE), polyethylene amine (PEI), dimethyl-aminomethyl (DMAE), andtrimethylaminoethyl (TMAE).

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the cation exchangechromatography resin is any one selected from the group consisting ofmethylsulfonate (S), sulfopropyl (SP), carboxymethyl (CM), sulfoethyl(SE), and polyaspartic acid.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the hydrophobicchromatography resin is any one selected from the group consisting ofphenyl, octyl, (iso)propyl, butyl, and ethyl.

Still another specific embodiment of the present invention provides thepreparation method in which the functional group of the affinitychromatography resin is any one selected from the group consisting ofprotein A, heparin, blue, benzamidine, metal ions (cobalt, nickel, andcopper), an antibody to a part or the entirety of constitutingcomponents of the protein complex, in which both ends of thenon-peptidyl polymer are respectively conjugated to the immunoglobulinFc region and the physiologically active polypeptide.

Still another specific embodiment of the present invention provides thepreparation method in which the resin of the size exclusionchromatography is selected from the group consisting of Superdex,Sephacryl, Superpose, and Sephadex.

Still another specific embodiment of the present invention provides thepreparation method in which step (b) is to isolate the protein complexin which the non-peptidyl polymer and an immunoglobulin Fc region,constituting a protein complex, are linked through the N-terminus of theimmunoglobulin Fc region.

Still another aspect of the present invention provides a method ofpreparing the position-specific protein complex, the method comprising:

(a′) preparing a conjugate by linking one end of the non-peptidylpolymer to the immunoglobulin Fc region or the physiologically activepolypeptide by a covalent bond, which is performed in a pH conditionfrom 4.0 to 9.0;

(b′) isolating the conjugate prepared in step (a′) and linking the otherend of the non-peptidyl polymer of the isolated conjugate to the otherof the immunoglobulin Fc region and the physiologically activepolypeptide by a covalent bond, which is performed in a pH conditionfrom 4.0 to 9.0; and (c′) isolating the protein complex, essentiallyincluding the covalently linked physiologically active polypeptide,non-peptidyl polymer, and immunoglobulin Fc region prepared in step(b′), in which the non-peptidyl polymer is linked to the N-terminus ofthe immunoglobulin Fc fragment.

In particular, an important condition for a reaction rate in bindingbetween the non-peptidyl polymer and the N-terminus of theimmunoglobulin Fc region is pH, and the site-specific binding may occurwell at a pH value below neutral pH, that is, below pH 7.0.

The linking of the non-peptidyl polymer to the N-terminus of theimmunoglobulin Fc region is performed at a pH value below neutral pH,but suitably performed at a weak acidic to acidic pH which does notdenature a tertiary structure or activity of the protein, but is notlimited thereto. As a non-limiting example, the immunoglobulin Fc regionused in the present invention has an amino acid sequence of SEQ ID NO:2, and it was confirmed to have N-terminal specificity at a weak basiccondition of about pH 8.2 (Example 5).

That is, when a general immunoglobulin Fc region is used, the reactionrate of specific binding of N-terminal of the immunoglobulin Fc regionand the non-peptidyl polymer is increased at a pH below neutral pH.However, when an immunoglobulin Fc region mutated to have a lower pHsensitivity is used, the reaction rate of the binding may not berestricted to the condition.

Still another aspect of the present invention provides a method ofpreparing the protein complex, the method comprising:

(a′) preparing a conjugate by linking one end of the non-peptidylpolymer to any one of the immunoglobulin Fc region and thephysiologically active polypeptide by a covalent bond, in which thereaction mole ratio between the physiologically active polypeptide andthe non-peptidyl polymer is in the range from 1:1 to 1:30, and thereaction mole ratio between the immunoglobulin Fc region and thenon-peptidyl polymer is in the range from 1:1 to 1:20, a reducing agentis contained in the range from 1 mM to 100 mM, and the reaction isperformed in the condition of pH from 4.0 to 9.0, at a temperature from4.0° C. to 25° C., and the reaction concentration of the immunoglobulinFc region or physiologically active polypeptide is in the range from 0.1mg/mL to 100 mg/mL;

(b′) isolating the conjugate prepared in step (a′) and linking the otherend of the non-peptidyl polymer of the isolated conjugate to the otherof the immunoglobulin Fc region and the physiologically activepolypeptide by a covalent bond, in which the reaction mole ratio betweenthe conjugate and the immunoglobulin Fc region or the physiologicallyactive polypeptide is in the range from 1:0.1 to 1:20, a reducing agentis contained in the range from 1 mM to 100 mM, and the reaction isperformed in the condition of pH from 4.0 to 9.0, at a temperature from4.0° C. to 25° C., and the reaction concentration of the immunoglobulinFc region or physiologically active polypeptide is in the range from 0.1mg/mL to 100 mg/mL; and

(c′) isolating the protein complex, essentially comprising thecovalently linked physiologically active polypeptide, non-peptidylpolymer, and immunoglobulin Fc region prepared in step (b′), in whichthe non-peptidyl polymer is linked to the N-terminus of theimmunoglobulin Fc fragment.

Still another specific embodiment of the present invention provides amethod for preparing the protein complex with N-terminal selectivity of90% or higher.

Still another aspect of the present invention provides a pharmaceuticalcomposition for improving in vivo duration and stability of thephysiologically active polypeptide comprising the protein complex as anactive ingredient.

A specific embodiment of the present invention provides a compositioncomprising the protein complex in an amount of 90% or higher.

Still another aspect of the present invention is a pharmaceuticalcontainer containing the preparation for delivery of the presentcomposition. Exemplary pharmaceutical containers include an injector, asyringe, vial, infusion bottle, ampoule or carpoule, for example, asyringe equipped with a needle protection system or a carpoule within aninjection pen. According to one embodiment, the present inventionprovides an injector that includes a container having a wall with aninterior surface and a seal assembly with an interior surface, theinterior surfaces of the wall and the seal assembly defining a closedsterile reservoir filled with a drug product.

The injector may also include a fluid delivery system comprising aclean, unsheathed, rigid container needle having a point disposed onlypartially through the seal assembly in a storage state, and disposedthrough the interior surface of the seal assembly into the sterilereservoir in a delivery state. Further, the injection may include anactuator that is adapted to move the container needle from the storagestate to the delivery state. In one embodiment, the wall of thecontainer may be a rigid wall or a flexible wall, and the seal assemblymay be a flexible unitary wall having an interior surface that definesthe interior surface of the seal assembly. The flexible unitary wall maydefine a septum disposed across the opening and fixedly attached to thewall of the container. Alternatively, the wall of the container maydefine a bore, and the unitary flexible wall may define a stopper thatis moveable along the bore. In such a case, the wall of the containermay define a closed end opposite the stopper and an open end in whichthe stopper is disposed. As a further alternative, the wall of thecontainer may define a bore with an opening in fluid communication witha first end of the bore, and the unitary flexible wall defines a septumdisposed across the opening and fixedly attached to the wall of thecontainer, the container further comprising a stopper that is disposedwithin a second end of the bore and is moveable along the bore.

Still another aspect of the present invention is directed to methods oftreating, improving, or preventing neurological disorders in patients inneed thereof. Such neurological disorders can be associated with chronicspinal cord injuries, Parkinson's disease, or neurodevelopmentaldisabilities such as cerebral palsy. The method comprises administeringtherapeutically effective doses to patients in need in connection to theneurological disorder and management thereof.

Still another aspect of the present invention provides for the use ofthe instant composition as set forth above in treatment relating to bonemarrow transplantation. The method comprises administeringtherapeutically effective doses to patients in need in connection withbone marrow transplantation and mobilization of stem cells.

Definitions

As used herein, the term “protein complex” or “complex” refers to astructure in which at least one physiologically active polypeptide, atleast one non-peptidyl polymer having a reactive group at both endsthereof, and at least one immunoglobulin Fc region are linked to eachother via a covalent bond. Further, a structure in which only twomolecules selected from the physiologically active polypeptide, thenon-peptidyl polymer, and the immunoglobulin Fc region are linked toeach other via a covalent bond is called “conjugate” in order todistinguish it from the “complex.”

The protein complex of the present invention may be a protein complex inwhich the PEG is linked to the modified G-CSF and the immunoglobulin Fcregion through reactive groups at both ends thereof by a covalent bond,respectively.

As used herein, the term “physiologically active polypeptide,”“physiologically active protein,” “active protein,” or “protein drug”refers to a polypeptide or a protein having some kind of antagonisticactivity to a physiological event in vivo, and these terms may be usedinterchangeably.

As used herein, the term “non-peptidyl polymer” refers to abiocompatible polymer including two or more repeating units which arelinked to each other by any covalent bond excluding a peptide bond, butis not limited thereto.

As used herein, the term “immunoglobulin Fc region” refers to a regionof an immunoglobulin molecule, except for the variable regions of theheavy and light chains, the heavy-chain constant region 1 (CH1) and thelight chain constant region 1 (CL1) of an immunoglobulin. Theimmunoglobulin Fc region may further include a hinge region at theheavy-chain constant region. In particular, the immunoglobulin Fc regionof the present invention may be a fragment including a part or all ofthe Fc region, and in the present invention, the immunoglobulin Fcregion may be used interchangeably with an immunoglobulin fragment.

A native Fc has a sugar chain at position Asn297 of heavy-chain constantregion 1, but E. coli-derived recombinant Fc is expressed as anaglycosylated form. The removal of sugar chains from Fc results in adecrease in binding affinity of Fc gamma receptors 1, 2, and 3 andcomplement (clq) to heavy-chain constant region 1, leading to a decreaseor loss in antibody-dependent cell-mediated cytotoxicity orcomplement-dependent cytotoxicity.

As used herein, the term “immunoglobulin constant region” may refer toan Fc fragment including heavy-chain constant region 2 (CH2) andheavy-chain constant region 3 (CH3) (or containing heavy-chain constantregion 4 (CH4)), except for the variable regions of the heavy and lightchains, the heavy-chain constant region 1 (CHI) and the light chainconstant region (CL) of an immunoglobulin, and may further include ahinge region at the heavy chain constant region. Further, theimmunoglobulin constant region of the present invention may be anextended immunoglobulin constant region including a part or all of theFc region including the heavy-chain constant region 1 (CH1) and/or thelight chain constant region (CL), except for the variable regions of theheavy and light chains of an immunoglobulin, as long as it has aphysiological function substantially similar to or better than thenative protein.

Meanwhile, the immunoglobulin constant region may originate from humansor animals, such as cows, goats, pigs, mice, rabbits, hamsters, rats,guinea pigs, etc., and may preferably be of human origin. In addition,the immunoglobulin constant region may be selected from constant regionsderived from IgG, IgA, IgD, IgE, IgM, or combinations or hybridsthereof, preferably, derived from IgG or IgM, which are the mostabundant thereof in human blood, and most preferably, derived from IgG,which is known to improve the half-life of ligand-binding proteins. Inthe present invention, the immunoglobulin Fc region may be a dimer ormultimer consisting of single-chain immunoglobulins of domains of thesame origin.

As used herein, the term “combination” means that polypeptides encodingsingle-chain immunoglobulin constant regions (preferably Fc regions) ofthe same origin are linked to a single-chain polypeptide of a differentorigin to form a dimer or multimer. That is, a dimer or a multimer maybe prepared from two or more fragments selected from the groupconsisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgEFc.

As used herein, the term “hybrid” means that sequences encoding two ormore immunoglobulin constant regions of different origins are present ina single-chain of an immunoglobulin constant region (preferably, an Fcregion). In the present invention, various hybrid forms are possible.For example, the hybrid domain may be composed of one to four domainsselected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc,IgM Fc, IgA Fc, IgE Fc, and IgD Fc, and may further include a hingeregion.

IgG may be divided into the IgG1, IgG2, IgG3, and IgG4 subclasses, andthe present invention may include combinations or hybrids thereof.Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fcregion of IgG4 rarely having effector functions such as complementdependent cytotoxicity (CDC).

The immunoglobulin constant region may have the glycosylated form to thesame extent as, or to a greater or lesser extent than the native form ormay be the deglycosylated form. Increased or decreased glycosylation ordeglycosylation of the immunoglobulin constant region may be achieved bytypical methods, for example, by using a chemical method, an enzymaticmethod or a genetic engineering method using microorganisms. Herein,when deglycosylated, the complement (Clq) binding to an immunoglobulinconstant region becomes significantly decreased, and antibody-dependentcytotoxicity or complement-dependent cytotoxicity is reduced or removed,thereby not inducing unnecessary immune responses in vivo. In thiscontext, deglycosylated or aglycosylated immunoglobulin constant regionsare more consistent with the purpose of drug carriers. Accordingly, theimmunoglobulin Fc region may be more specifically an aglycosylated Fcregion derived from human IgG4, that is, a human IgG4-derivedaglycosylated Fc region. The human-derived Fc region is more preferablethan a non-human derived Fc region, which may act as an antigen in thehuman body and cause undesirable immune responses such as the productionof a new antibody against the antigen.

Further, the immunoglobulin constant region of the present inventionincludes not only the native amino acid sequence but also sequencederivatives (mutants) thereof. The amino acid sequence derivative meansthat it has an amino acid sequence different from the wild-type aminoacid sequence as a result of deletion, insertion, conserved ornon-conserved substitution of one or more amino acid residues, or acombination thereof. For instance, amino acid residues at positions 214to 238, 297 to 299, 318 to 322, or 327 to 331 in IgG Fc, known to beimportant for linkage, may be used as the sites suitable formodification. Various derivatives, such as those prepared by removingthe sites capable of forming disulfide bonds, removing severalN-terminal amino acids from native Fc, or adding methionine to theN-terminus of native Fc, may be used. In addition, complement fixationsites, e.g., Clq fixation sites, or ADCC sites, may be eliminated toremove the effector function. The techniques of preparing the sequencederivatives of the immunoglobulin constant region are disclosed inInternational Patent Publication Nos. WO 97/34631 and WO 96/32478.

Amino acid substitutions in a protein or peptide molecule that do notalter the activity of the molecule are well known in the art (H.Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost common substitutions occur between amino acid residues Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, AlaJGly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal, Ala/Glu, andAsp/Gly, in both directions. Optionally, amino acids may be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, or the like.

The above-described immunoglobulin constant region derivative may be aderivative which has a biological activity equivalent to that of theimmunoglobulin constant region of the present invention, but hasincreased structural stability of the immunoglobulin constant regionagainst heat, pH, etc. Further, the immunoglobulin constant region maybe obtained from a native type isolated from humans or animals such ascows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., ormay be their recombinants or derivatives obtained from transformedanimal cells or microorganisms. Herein, they may be obtained from anative immunoglobulin by isolating whole immunoglobulins from human oranimal organisms and treating them with a proteolytic enzyme. Papaindigests the native immunoglobulin into Fab and Fc regions, and pepsintreatment results in the production of pF′c and F(ab)2 fragments. Thesefragments may be subjected, for example, to size exclusionchromatography to isolate Fc or pF′c.

Preferably, a human-derived immunoglobulin constant region may be arecombinant immunoglobulin constant region that is obtained from amicroorganism.

The protein complex of the present invention may include one or more ofa unit structure of a [physiologically active polypeptide/non-peptidylpolymer/immunoglobulin Fc region], in which all components may be linkedin a linear form by a covalent bond. The non-peptidyl polymer may have areactive group at both ends thereof and is linked to the physiologicallyactive polypeptide and the immunoglobulin Fc region through the reactivegroup by a covalent bond, respectively. That is, at least one conjugateof the physiologically active polypeptide and the non-peptidyl polymeris linked to one immunoglobulin Fc region by a covalent bond, therebyforming a monomer, dimer, or multimer of the physiologically activepolypeptide, which is mediated by the immunoglobulin Fc region.Therefore, an increase in vivo activity and stability may be moreeffectively achieved.

The reactive group at both ends of the non-peptidyl polymer ispreferably selected from the group consisting of a reactive aldehydegroup, a propionaldehyde group, a butyraldehyde group, a maleimidegroup, and a succinimide derivative. The succinimide derivative may behydroxy succinimidyl, succinimidyl carboxymethyl, succinimidyl valerate,succinimidyl methyl butanoate, succinimidyl methyl propionate,succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide,or succinimidyl carbonate. In particular, when the non-peptidyl polymerhas a reactive aldehyde group at both ends, it is effective in linkingboth of the ends with the physiologically active polypeptide and theimmunoglobulin with minimal non-specific reactions. A final productgenerated by reductive alkylation by an aldehyde bond is much morestable than when linked by an amide bond.

The reactive groups at both ends of the non-peptidyl polymer of thepresent invention may be the same as or different from each other. Thenon-peptide polymer may possess aldehyde reactive groups at both ends,or it may possess an aldehyde group at one end and a maleimide reactivegroup at the other end, or an aldehyde group at one end and asuccinimide reactive group at the other end, but is not limited thereto.

For example, the non-peptide polymer may possess a maleimide group atone end and an aldehyde group, a propionaldehyde group, or abutyraldehyde group at the other end. Also, the non-peptide polymer maypossess a succinimidyl group at one end and a propionaldehyde group, ora butyraldehyde group at the other end. When a polyethylene glycolhaving a reactive hydroxy group at both ends thereof is used as thenon-peptidyl polymer, the hydroxy group may be activated to variousreactive groups by known chemical reactions, or a commercially availablepolyethylene glycol having a modified reactive group may be used so asto prepare the protein complex of the present invention.

When the physiologically active polypeptide and the immunoglobulin Fcregion are linked via the non-peptidyl polymer, each of both of the endsof the non-peptidyl polymer may bind to the N-terminus of theimmunoglobulin Fc region and the N-terminus (amino terminus), C-terminus(carboxy terminus), or free reactive group of a lysine residue, ahistidine residue, or a cysteine residue of the physiologically activepolypeptide.

As used herein, the term “N-terminus” refers to an N-terminus of apeptide, which is a site to which a linker including a non-peptidylpolymer can be conjugated for the purpose of the present invention.Examples of the N-terminus may include not only amino acid residues atthe distal end of the N-terminus, but hut also amino acid residues nearthe N-terminus, but are not limited thereto. Specifically, the 1st tothe 20th amino acid residues from the distal end may be included.

The non-peptidyl polymer of the present invention may be selected fromthe group consisting of polyethylene glycol, polypropylene glycol,copolymers of ethylene glycol and propylene glycol, polyoxyethylatedpolyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethylether, biodegradable polymers such as PLA (polylactic acid) and PLGA(polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid,and combinations thereof, and specifically, polyethylene glycol, but isnot limited thereto. Also, derivatives thereof well known in the art andeasily prepared within the skill of the art are included in thenon-peptidyl polymer of the present invention. The non-peptidyl polymermay have a molecular weight in the range of 1 kDa to 100 kDa, andspecifically 1 kDa to 20 kDa.

The physiologically active polypeptide of the present invention may beexemplified by various physiologically active polypeptides such ashormones, cytokines, interleukins, interleukin-binding proteins,enzymes, antibodies, growth factors, transcription factors, bloodfactors, vaccines, structural proteins, ligand proteins or receptors,cell surface antigens, receptor antagonists, and derivatives or analogsthereof.

Specifically, the physiologically active polypeptide includes humangrowth hormones, growth hormone-releasing hormones, growthhormone-releasing peptides, interferons and interferon receptors (e.g.,interferon-alpha, -beta, and -gamma, soluble type I interferonreceptors), colony-stimulating factors, interleukins (e.g.,interleukin-1, -2, -3, -4, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15,-16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29.-30. Etc.), and interleukin receptors (e.g; IL-1 receptor. IL-4receptor, etc.), enzymes (e.g., glucocerebrosidase,iduronate-2-sulfatase, alpha-galactosidase-A, agalsidase alpha,beta,alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamatedecarboxylase, imiglucerase, lipase, uricase, platelet-activating factoracetylhydrolase, neutral endopeptidase, myeloperoxidase, etc.),interleukin- and cytokine-binding proteins (e.g., IL-18 bp, TNF-bindingprotein, etc.), macrophage-activating factors, macrophage peptides,B-cell factors, T-cell factors, protein A, allergy inhibitors, cellnecrosis glycoproteins, immunotoxins, lymphotoxins, tumor necrosisfactor, tumor suppressors, transforming growth factor, alpha-1anti-trypsin, albumin, alpha-lactalbumin, apolipoprotein-E,erythropoietin, glycosylated crythropoictin, an-giopoietins, hemoglobin,thrombin, thrombin receptors activating peptides, throm-bomodulin, bloodfactors VII, VIIa, VIII, IX, and XIII, plasminogen activators,fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C,C-reactive protein, renin inhibitor, collagenase inhibitor, superoxidedismutase, leptin, platelet-derived growth factor, epithelial growthfactor, epidermal growth factor, angiostatin, angiotensin, bone growthfactor, bone-stimulating protein, calcitonin, insulin, oxyntomodulin,glucagon, glucagon derivatives, glucagon-like peptides, exendins(Exendin4), atriopeptin, cartilage-inducing factor, elcatonin,connective tissue-activating factor, tissue factor pathway inhibitor,follicle-stimulating hormone, luteinizing hormone, luteinizinghormone-releasing hormone, nerve growth factors (e.g., nerve growthfactor, cilliary neurotrophic factor, axogenesis factor-1,brain-natriuretic peptide, glial-derived neu-rotrophic factor, netrin,neutrophil inhibitor factor, neurotrophic factor, neurturin, etc.),parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growthfactor, adrenocortical hormone, glucagon, cholecystokinin, pancreaticpolypeptide, gastrin-releasing peptide, corticotrophin-releasing factor,thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin,receptors (e.g., TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor,B-cell-activating factor receptor, etc.), receptor antagonists (e.g.,IL1-Ra, etc.), cell surface antigens (e.g., CD 2, 3, 4, 5, 7, 11a, 11b,18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab′,F(ab′)2, and Fd), and virus-derived vaccine antigens.

Specifically, the physiologically active polypeptide of the presentinvention may be granulocyte colony-stimulating factor, erythropoietin,or modified versions thereof. In the preferred embodiment, thepolypeptide is G-CSF.

In the present invention, the antibody fragment may be Fab, Fab′,F(ab′), Fd, or scFv having an ability to bind to a specific antigen, andpreferably, Fab.′ The Fab fragments include the variable domain (VL) andconstant domain (CL) of the light chain and the variable domain (VH) andthe first constant domain (CH1) of the heavy chain. The Fab′ fragmentsdiffer from the Fab fragments in terms of the addition of several aminoacid residues including one or more cysteine residues from the hingeregion at the carboxyl terminus of the CH1 domain. The Fd fragments arefragments consisting of only the VH and CH1 domains, and the F(ab′)2fragments are produced by binding of two molecules of Fab′ fragments byeither disulfide bonding or a chemical reaction. The scFv fragment is asingle polypeptide chain, in which only VL and VH domains are linked toeach other by a peptide linker

Further, the protein complex of the present invention may be used in thedevelopment of long-acting protein formulations of animal growth hormonesuch as bovine growth hormone or porcine growth hormone, and long-actingprotein formulations for treatment or prevention of animal disease, suchas interferon.

Another aspect of the present invention provides a method of preparingthe protein complex of the present invention. In particular, the presentinvention provides a method of preparing a position-specific proteincomplex, the method comprising: (a) preparing a protein complex bylinking at least one non-peptidyl polymer having a reactive group atboth ends, at least one physiologically active polypeptide, and at leastone immunoglobulin Fc region by a covalent bond, and (b) isolating theprotein complex, essentially including the covalently linkedphysiologically active polypeptide, non-peptidyl polymer, andimmunoglobulin Fc region prepared in step (a), in which the non-peptidylpolymer is linked to the N-terminus of the immunoglobulin Fc fragment.

The immunoglobulin Fc region of the present invention may be in the formof a dimer, or in the form of a homodimer or heterodimer. Therefore, theimmunoglobulin Fc region constituting the protein complex of the presentinvention may include one or two or more of an N-terminus. Thus, theimmunoglobulin Fc region may be linked to at least one non-peptidylpolymer via the N-terminus. In particular, the immunoglobulin Fc regionof the present invention may be in the form of a homodimer, andtherefore, linked to one or two non-peptidyl polymers via twoN-terminals included in the homodimer of the immunoglobulin Fc region.In this regard, the non-peptidyl polymers may bind to thephysiologically active polypeptides, respectively, thereby forming theprotein complex.

Accordingly, the protein complex of the present invention may beprepared by linking one or two or more of the non-peptidyl polymer, oneor two or more of the physiologically active polypeptide, and one or twoor more of the immunoglobulin Fc region via a covalent bond.

In step (a), the covalent bonds between the three components may occursequentially or at the same time. For example, when the physiologicallyactive polypeptide and the immunoglobulin Fc region are linked to bothends of the non-peptidyl polymer, respectively, any one of thephysiologically active polypeptide and the immunoglobulin Fc region maybe first linked to one end of the non-peptidyl polymer, and then theother may be linked to the other end of the non-peptidyl polymer. Thismethod is advantageous in that production of by-products other than thedesired protein complex is minimized, and the protein complex isprepared in high purity.

Therefore, step (a) may comprise:

(i) linking a specific site of the immunoglobulin Fc region or thephysiologically active polypeptide to one end of the non-peptidylpolymer via a covalent bond;

(ii) homogeneously isolating a conjugate from the reaction mixture, inwhich the conjugate is prepared by linking the specific site of theimmunoglobulin Fc region or the physiologically active polypeptide tothe non-peptidyl polymer; and

(iii) producing a protein complex by linking the physiologically activepolypeptide or the specific site of the immunoglobulin Fc region to theother end of the non-peptidyl polymer of the isolated conjugate.

Meanwhile, in the present invention, step (a) includes (a1) preparing aconjugate by linking one end of the non-peptidyl polymer to any one ofthe immunoglobulin Fc region and the physiologically active polypeptideby a covalent bond; and (a2) isolating the conjugate prepared in step(a1) and linking the other end of the non-peptidyl polymer of theisolated conjugate to the other of the physiologically activepolypeptide and the immunoglobulin Fc region by a covalent bond.

Specifically, step (a) may comprise (a1′) preparing a conjugate bylinking one end of the non-peptidyl polymer to the immunoglobulin Fcregion by a covalent bond; and (a2′) isolating the conjugate prepared instep (a1′) and linking the other end of the non-peptidyl polymer of theisolated conjugate to the physiologically active polypeptide by acovalent bond.

Alternatively, step (a) may include (a1″) preparing a conjugate bylinking one end of the non-peptidyl polymer to the physiologicallyactive polypeptide by a covalent bond; and (a2″) isolating the conjugateprepared in step (a1″) and linking the other end of the non-peptidylpolymer of the isolated conjugate to the immunoglobulin Fc region by acovalent bond.

In step (a1), (a1), or (a1″) of the present invention, the reaction moleratio between the physiologically active polypeptide and thenon-peptidyl polymer may be in the range from 1:1 to 1:30, and thereaction mole ratio between the immunoglobulin Fc region and thenon-peptidyl polymer may be in the range from 1:1 to 1:20.

Specifically, in step (a1), the reaction mole ratio between theimmunoglobulin Fc region and the non-peptidyl polymer may be in therange from 1:1 to 1:20, and in particular, in the range from 1:1 to1:15, 1:1 to 1:10, or 1:1 to 1:4. In step (a1″), the reaction mole ratiobetween the physiologically active polypeptide and the non-peptidylpolymer may be in the range from 1:1 to 1:30, and in particular, in therange from 1:1 to 1:15 or 1:1 to 1:10. A preparation yield and cost maybe optimized depending on the reaction mole ratio.

In the present invention, step (a1), (a1), or (a1″) may be performed ina pH condition from 4.0 to 9.0; step (a1), (a1), or (a1″) may beperformed at a temperature from 4.0° C. to 25° C.; in step (a1), (a1),or (a1″), the reaction concentration of the immunoglobulin Fc region orphysiologically active polypeptide may be in the range from 0.1 mg/mL to100 mg/mL.

In step (a2), (a2′), or (a2″) of the present invention, the reactionmole ratio between the conjugate and the immunoglobulin Fc region or thephysiologically active polypeptide may be in the range from 1:0.1 to1:20, and in particular, in the range from 1:0.2 to 1:10. Specifically,in step (a2′), the reaction mole ratio between the conjugate and thephysiologically active polypeptide may be in the range from 1:0.1 to1:20, and in step (a2″), the reaction mole ratio between the conjugateand the immunoglobulin Fc region may be in the range from 1:0.1 to 1:20.A preparation yield and cost may be optimized depending on the reactionmole ratio.

In the present invention, step (a2), (a2′), or (a2″) may be performed ina pH condition from 4.0 to 9.0; step (a2), (a2′), or (a2″) may beperformed at a temperature from 4.0° C. to 25° C.; in step (a2), (a2′),or (a2″), the reaction concentration of the immunoglobulin Fc region orphysiologically active polypeptide may be in the range from 0.1 mg/mL to100 mg/mL.

Meanwhile, the preparation method of the present invention may be amethod of preparing a position-specific protein complex, including (a′)preparing a conjugate by linking one end of the non-peptidyl polymer toany one of the immunoglobulin Fc region and the physiologically activepolypeptide by a covalent bond, in which the reaction mole ratio betweenthe physiologically active polypeptide and the non-peptidyl polymer isin the range from 1:1 to 1:30, the reaction mole ratio between theimmunoglobulin Fc region and the non-peptidyl polymer is in the rangefrom 1:1 to 1:20, a reducing agent is contained in the range from 1 mMto 100 mM, the reaction is performed in the condition of pH from 4.0 to9.0, at a temperature from 4.0° C. to 25° C., and the reactionconcentration of the immunoglobulin Fc region or physiologically activepolypeptide is in the range from 0.1 mg/mL to 100 mg/mL;

(b′) isolating the conjugate prepared in step (a′) and linking the otherend of the non-peptidyl polymer of the isolated conjugate to the otherof the immunoglobulin Fc region and the physiologically activepolypeptide by a covalent bond, in which the reaction mole ratio betweenthe conjugate and the immunoglobulin Fc region or the physiologicallyactive polypeptide is in the range from 1:0.1 to 1:20, a reducing agentis contained in the range from 1 mM to 100 mM, the reaction is performedin the condition of pH from 4.0 to 9.0, at a temperature from 0° C. to25° C., and the concentration of the immunoglobulin Fc region orphysiologically active polypeptide is in the range from 0.1 mg/mL to 100mg/mL; and

(c′) isolating the protein complex, essentially including the covalentlylinked physiologically active polypeptide, non-peptidyl polymer, andimmunoglobulin Fc region prepared in step (b′), in which thenon-peptidyl polymer is linked to the N-terminus of the immunoglobulinFc fragment, but is not limited thereto.

The reactions in step (a1), step (a1), step (a1″), step (a2), step(a2′), and step (a2″) of the present invention may be performed in thepresence of a reducing agent, considering the type of the reactivegroups at both ends of the non-peptidyl polymer which participate in thereactions, if necessary. The reducing agent of the present invention maybe sodium cyanoborohydride (NaCNBH3), sodium borohydride, dimethylamineborate, or pyridine borate. In this regard, a concentration of thereducing agent (e.g., sodium cyanoborohydride), temperature and pH of areaction solution, and total concentrations of the physiologicallyactive polypeptide and the immunoglobulin Fc region participating in thereaction are important in terms of production yield and purity. Tomaximize the production of a high-purity homogeneous complex, variouscombinations of the conditions are needed. According to the feature ofthe physiologically active polypeptide to be prepared, variousconditions are possible, but not limited to, the reducing agent (e.g.,sodium cyanoborohydride) may be contained in the range from 1 mM to 100mM, the reaction solution may be at a temperature from 0° C. to 25° C.and in the condition of pH from 4.0 to 9.0, and the concentration of thereaction protein (concentration of the immunoglobulin Fc region orphysiologically active polypeptide included upon the reaction) may be inthe range from 5 mg/mL to 100 mg/mL.

Meanwhile, the separation of the conjugate in step (a2), step (a2′), andstep (a2″) may be performed, if necessary, by a method selected fromgeneral methods which are used in protein separation, considering theproperties such as purity, hydrophobicity, molecular weight, andelectrical charge which are required for the separated conjugate. Forexample, the separation may be performed by applying various knownmethods, including size exclusion chromatography, affinitychromatography, hydrophobic chromatography, or ion exchangechromatography, and if necessary, a plurality of different methods areused in combination to purify the conjugate with higher purity.

According to the features of the physiologically active polypeptide tobe prepared, various conditions are possible. However, in order toseparate the immunoglobulin Fc region or the physiologically activepolypeptide conjugate linked to the non-peptidyl polymer, size exclusionchromatography is generally performed. For further scale-up andseparation of isomers generated by binding of the non-peptidyl polymerat a position other than the desired position or a small amount ofdenatured forms generated during preparation, affinity chromatography,hydrophobic chromatography, or ion exchange chromatography may also beused.

In the present invention, step (b) may be performed, if necessary, by amethod selected from general methods which are used in proteinseparation, considering the properties such as hydrophobicity, molecularweight, and electrical charge, in order to finally purify a high-puritycomplex. For example, the separation may be performed by applyingvarious known methods, including size exclusion chromatography, affinitychromatography, hydrophobic chromatography, or ion exchangechromatography, and if necessary, a plurality of different methods areused in combination to purify the complex with higher purity. Accordingto the features of the desired complex consisting of the physiologicallyactive polypeptide, the non-peptidyl polymer, and the Fc constantregion, various separation conditions are possible. However, in order toseparate the complex in which the physiologically active polypeptide andthe immunoglobulin Fc region are respectively linked to both ends of thenon-peptidyl polymer, size exclusion chromatography is generallyperformed. For further scale-up and effective separation of isomers orside-reaction products generated by binding of the physiologicallyactive polypeptide or the immunoglobulin Fc region, and non-peptidylpolymer at a position other than the desired position, or a small amountof denatured forms generated during preparation, unreactedphysiologically active polypeptide, non-peptidyl polymer, andimmunoglobulin Fc region, hydrophobic chromatography, ion exchangechromatography, or affinity chromatography may be used in combination.In particular, hydrophobic chromatography and ion exchangechromatography may be used in combination, and a plurality ofhydrophobic chromatography or a plurality of ion exchange chromatographyis also possible. According to the complex to be prepared, ion exchangechromatography or hydrophobic chromatography may be used singly.

In the present invention, the ion exchange chromatography is to separatea protein by passing charged protein at a specific pH through a chargedion resin-immobilized chromatography column and separating the proteinby a difference in the migration rate of the protein, and it may beanion exchange chromatography or cation exchange chromatography.

The anion exchange chromatography is to use a cation resin, and afunctional group of the resin constituting the corresponding anionexchange chromatography may be any one selected from the groupconsisting of quaternary ammonium (Q), quaternary aminoethyl (QAE),diethylaminoethyl (DEAE), polyethylene amine (PEI), dimethyl-aminomethyl(DMAE), and trimethylaminoethyl (TMAE), but is not limited thereto.

Further, the cation exchange chromatography is to use an anion resin,and a functional group of the resin constituting the correspondingcation exchange chromatography may be any one selected from the groupconsisting of methylsulfonate (S), sulfopropyl (SP), carboxymethyl (CM),sulfoethyl (SE), and polyaspartic acid, but is not limited thereto.

In the present invention, a functional group of the resin constitutingthe hydrophobic chromatography may be any one selected from the groupconsisting of phenyl, octyl, (iso)propyl, butyl, and ethyl, but is notlimited thereto.

In the present invention, a functional group of the resin constitutingthe size exclusion chromatography may be any one selected from the groupconsisting of Superdex, Sephacryl, Superpose, and Sephadex, but is notlimited thereto.

Furthermore, the affinity chromatography in the present invention is toseparate a protein by a difference in the migration rate of the protein,which is caused by the interaction between the protein and a ligandcapable of interacting with the protein in a resin onto which the ligandis immobilized. A functional group of the resin constituting theaffinity chromatography may be any one selected from the groupconsisting of protein A, heparin, blue, benzamidine, metal ions (cobalt,nickel, and copper), and an antibody to a part or the entirety of theconstituting components of the to protein complex, in which both ends ofthe non-peptidyl polymer arc respectively conjugated to theimmunoglobulin Fc region and the physiologically active polypeptide, butis not limited thereto.

In the present invention, step (b) is to isolate the protein complex inwhich the non-peptidyl polymer and the immunoglobulin Fc region arelinked to each other via the N-terminus of the immunoglobulin Fc region.

Still another aspect of the present invention provides a method forpreparing a protein complex with N-terminal selectivity of 90% orhigher. Specifically, the protein complex prepared by the method of thepresent invention may be one, in which one end of the non-peptidylpolymer may be linked to the N-terminus of the immunoglobulin Fc regionwith N-terminal selectivity of 90% or higher, more specifically 95% orhigher, even more specifically 98% or higher, and yet even morespecifically 99% or higher, but is not limited thereto.

As used herein, the term “linking with N-terminal selectivity of 90% orhigher” means that, in 90% or more of the protein complex prepared bypurification of the protein complex fractions obtained by a series ofreactions according to the present invention, the non-peptidyl polymeris linked to the N-terminus of the Fc region in a position-specificmanner. As used herein, the term “90% or higher” may refer to v/v, w/w,and peak/peak, but is not limited to a particular unit. The yield of theprotein complex comprising the non-peptidyl polymer linked to theN-terminus of the Fc region in a position-specific manner may vary byreaction conditions, a reactor of the non-peptidyl polymer, etc.

In Examples of the present invention, it was confirmed that a proteincomplex with N-terminal selectivity of 90% or higher can be prepared bythe method according to the present invention, via preparation ofvarious physiologically active polypeptides, non-peptidyl polymers, andFc complexes.

The pharmaceutical composition may comprise a protein complex, whichincludes the physiologically active polypeptide-non-peptidylpolymer-N-terminus of an immunoglobulin Fc region, in an amount of 90%or higher, more specifically 95% or higher, even more specifically 98%or higher, and yet even more specifically 99% or higher, but is notlimited thereto. As used herein, the term “90% or higher” may refer tov/v, w/w, and peak/peak, but is not limited to a particular unit.

The pharmaceutical composition may further include a pharmaceuticallyacceptable excipient.

The pharmaceutical composition of the present invention may beadministered via various routes including oral, percutaneous,subcutaneous, intravenous, and intramuscular routes, preferably, in theform of an injectable formulation. Further, the pharmaceuticalcomposition of the present invention may be formulated by a method knownin the art in order to provide rapid, long-lasting, or delayed releaseof the active ingredient after administration thereof to a mammal. Theformulation may be a tablet, a pill, a powder, a sachet, an elixir, asuspension, an emulsion, a solution, a syrup, an aerosol, a soft or hardgelatin capsule, a sterile injectable solution, or a sterile powder.Examples of suitable carriers, excipients, and diluents may includelactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate,calcium silicate, cellulose, methyl cellulose, microcrystallinecellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Thepharmaceutical composition may further include a filler, ananticoagulant, a lubricant, a wetting agent, a flavoring agent, anemulsifying agent, a preservative, etc.

A practical administration dose of the protein complex of the presentinvention may be determined by several related factors including thetypes of diseases to be treated, administration routes, the patient'sage, gender, weight, and severity of the illness, as well as by thetypes of the physiologically active polypeptide as an active component.Since the protein complex of the present invention has excellent bloodduration and in vivo potency, it can remarkably reduce theadministration dose and frequency of a peptide drug, including theprotein complex of the present invention.

Still another aspect of the present invention provides a population ofprotein complexes, including the protein complex prepared according tothe above method in an amount of 90% or higher. As used herein, theterms “population of complex”, and “population” may be usedinterchangeably, and they refer to a group of protein complexesincluding protein complexes, in which a non-peptidyl polymer is linkedto the N-terminus of an Fc region, and/or protein complexes, in which anon-peptidyl polymer is linked to a region other than the N-terminus ofan Fc region.

The population may include only the protein complexes, in which anon-peptidyl polymer is linked to the N-terminus of an Fc region, or theprotein complexes, in which a non-peptidyl polymer is linked to a regionother than the N-terminus of an Fc region. Specifically, the percentageof the protein complexes, in which a non-peptidyl polymer is linked to aregion other than the N-terminus of an Fc region, included in thepopulation may be 90% or higher, more specifically 95% or higher, evenmore specifically 98% or higher, and yet even more specifically 99% orhigher, but is not limited thereto. As used herein, the term “90% orhigher” may refer to v/v, w/w, and peak/peak, but is not limited to aparticular unit.

For the purpose of the present invention, the population may refer to apopulation with an increased percentage of the protein complexes, inwhich a non-peptidyl polymer is linked to a region other than theN-terminus of an Fc region, by removing impurities, unreacted materials,etc., from the protein complexes prepared thereof. Additionally, thepopulation may refer to one which was prepared by a method for preparingprotein complexes with N-terminal selectivity of 90% or higher, but isnot limited thereto. The population may be efficiently purified by themethod of the present invention.

The present invention is particularly directed to the use of theabove-described protein complexes in preventing, alleviating, ortreating patient in need thereof having a need in increasing their whiteblood cell production, count, or are in need of increasing stem cellproduction by administering to the patient a therapeutically effectiveamount of a protein complex comprising a modified humangranulocyte-colony stimulating factor (hG-CSF) covalently linked to animmunoglobulin Fc region via a non-peptidyl polymer, wherein thenon-peptidyl polymer is site-specifically linked to an N-terminus of theimmunoglobulin Fc region, and the modified hG-CSF comprisessubstitutions in at least one of Cys17 and Pro65. Such methodologies mayor may not be in combination with chemotherapeutic agents or regimensincluding docetaxel, doxorubicin, cyclophosphamide (TAC); dose-densedoxorubicin plus cyclophosphamide (AC), with or without subsequentweekly or semiweekly paclitaxel; and docetaxel plus cyclophosphamide(TC). Regardless, the methodologies described in this invention providessuperior clinical and side effect outcomes for patients receiving such aregimen. In preferred embodiments, EFLAPEGRASTIM is administered at 13.2mg/0.6 mL (containing 3.6 mg G-CSF) fixed dose to the patient withinabout 26 hours, 24 hours, 22 hours, 18 hours, 12 hours, 6 hours, about 5hours, 2 hours, 1 hour or half an hour of the completion ofchemotherapy. In one embodiment, TC is administered on Day 1 of eachcycle intravenously (IV). Accordingly, Docetaxel is administered at 75mg/m² IV infusion and (ii) Cyclophosphamide is administered at 600 mg/m²IV infusion. Each treatment cycle is 21 days, with up to a maximum of 4cycles of chemotherapy. To begin full-dose chemotherapy on Day 1 of anycycle (Day 22 of the previous cycle), patients must show ANC ≥1.5×10⁹/Land a platelet count ≥100×10⁹/L. In other embodiments, EFLAPEGRASTIM maybe administered on Day 2 of each cycle, approximately 24 hours (±2hours) after TC chemotherapy.

Examples provided here are for illustrative purposes only, and theinvention is not intended to be limited by these Examples.

EXAMPLES Example 1: Preparation of Complex of Interferon Alpha(IFNu)-PEG-N-Terminus Region of

Immunoglobulin Fc

1-1. Preparation of 1FNa-PEG Conjugate

ALD-PEG-ALD (IDB, Korea), which is polyethylene glycol (PEG) having amolecular weight of 3.4 kDa and aldehyde reactive groups at both endsthereof, was added to 5 mg/mL of human interferon alpha-2b (hIFNa-2b,molecular weight: 19 kDa) dissolved in 100 mM phosphate buffer at amolar ratio of hIFNa:PEG of 1:5 to 1:10. A reducing agent, sodiumcyanoborohydride (NaBH₃CN, Sigma) was added thereto at a finalconcentration of 20 mM and allowed to react at 4° C. to 8° C. under slowstirring for about 1 hour. To obtain a conjugate in which PEG isselectively linked to the amino terminus of interferon alpha and PEG andinterferon alpha are linked to each other at a ratio of 1:1, thereaction mixture was subjected to SP HP (GE healthcare, USA) anionexchange chromatography to purify an IFNot-PEG conjugate with highpurity.

1-2. Preparation of IFNa-PEG-Fc Complex

In order to link the IFNa-PEG conjugate purified in Example 1-1 to theN-terminal proline residue of immunoglobulin Fc, the immunoglobulin Fcfragment was added and reacted at a molar ratio of IFNa-PEGconjugate:immunoglobulin Fc of 1:1 to 1:4. The reaction solution wasprepared as 100 mM phosphate buffer (pH 5.5 to 6.5), and sodiumcyanoborohydride (NaBH₃CN, Sigma) was added as a reducing agent at afinal concentration of 20 mM to 50 mM. The reaction was allowed at 4° C.to 8° C. for about 12 hours to 16 hours under slow stirring.

1-3. Isolation and Purification of IFNa-PEG-Fc Complex

In order to remove unreacted materials and by-products after the bindingreaction of Example 1-2 and to purify the IFNa-PEG-Fc protein complexthus produced, the reaction mixture was buffer-exchanged to 10 mM Tris(pH 7.5), and then passed through a Source Q (GE healthcare, USA) anionexchange chromatography column to remove unreacted Fc and to obtain anIFNa-PEG-Fc protein complex fraction. In detail, the reaction solutionwas applied to Source Q column equilibrated with 10 mM Tris (pH 7.5),and the column was subjected to isocratic solvent washing using 20 mMTris (pH 7.5) buffer solution containing 50 mM sodium chloride (NaCl) toremove impurities. Then, the IFNa-PEG-Fc protein complex was eluted witha concentration gradient of a buffer solution containing 150 mM sodiumchloride (NaCl). A small amount of unreacted Fc and interferon alphadimer were present as impurities in the obtained 1FNa-PEG-Fc proteincomplex fraction. In order to remove the impurities, Source iso (GEhealthcare, USA) hydrophobic chromatography was further performed. Indetail, Source iso (GE healthcare, USA) was equilibrated with a 20 mMpotassium phosphate (pH 6.0) buffer solution containing about 1.3 Mammonium sulfate, and then the purified IFNa-PEG-Fc protein complexfraction was applied thereto. Finally, a high-purity IFNa-PEG-Fc proteincomplex was purified with a linear concentration gradient of a 20 mMpotassium phosphate (pH 6.0) buffer solution. N-terminal selectivity ofthe Fc region of the prepared IFNa-PEG-Fc protein complex was examinedby peptide mapping, and the selectivity was found to be 90% or higher.

Example 2: Preparation of Human Granulocyte Colony Stimulating Factor(G-CSF)-PEG-Fc Complex

The ^(17,65S)G-CSF-PEG-Fc protein complex was prepared using aderivative (^(17,65)-G-CSF) prepared by substituting serine for theamino acids at positions 17 and 65 of the native G-CSF and thenpurified.

2-1. Preparation of ^(17,65S)G-CSF-PEG Conjugate

ALD-PEG-ALD (IDB, Korea), which is polyethylene glycol (PEG) having amolecular weight of 3.4 kDa and aldehyde reactive groups at both endsthereof, was added to 5 mg/mL of ^(17,65)S-G-CSF (molecular weight: 18kDa) dissolved in 100 mM phosphate buffer at a molar ratio of G-CSF:PEGof 1:5 to 1:10. A reducing agent, sodium cyanoborohydride (NaBH₃CN,Sigma), was added thereto at a final concentration of 20 mM and allowedto react at 4° C. to 8° C. under slow stirring for about 1 hour. Toobtain a conjugate in which PEG is selectively linked to the aminoterminus of human granulocyte colony stimulating factor and PEG andG-CSF are linked to each other at a ratio of 1:1, the reaction mixturewas subjected to SP HP (GE healthcare, USA) cation exchangechromatography to purify a ^(17,65S)G-CSF-PEG conjugate with a highpurity.

2-2. Preparation of ^(17,65)G-CSF-PEG-Fc Complex

In order to link the ^(17,65)G-CSF-PEG conjugate purified in Example 3-1to the N-terminus of immunoglobulin Fc, the immunoglobulin Fc fragmentwas added and reacted at a molar ratio of ^(17,65)Ser-G-CSF-PEGconjugate:immunoglobulin Fc of 1:1 to 1:4. The reaction solution wasprepared as a 100 mM phosphate buffer (pH 5.5 to 6.5), and sodiumcyanoborohydride (NaCNBH3, Sigma) was added as a reducing agent at afinal concentration of 20 mM. The reaction was allowed at 4° C. to 8° C.under slow stirring.

2-3. Isolation and Purification of ^(17,65)Ser-G-CSF-PEG-Fc (or^(17,65S)G-CSF-PEG-Fc) Complex

In order to remove unreacted materials and by-products after the bindingreaction of Example 3-2 and to purify the ^(17,65S)G-CSF-PEG-Fc proteincomplex thus produced, the reaction mixture was buffer-exchanged to 10mM Tris (pH 8.0) containing 2 M NaCl and then passed through a SourcePhenyl column. To remove impurities, the ^(17,65S)G-CSF-PEG-Fc proteincomplex was purified with a concentration gradient of 20 mM Tris (pH8.0) buffer solution. A small amount of unreacted immunoglobulin Fc and^(17,65)G-CSF dimer as impurities were present in the obtained^(17,65S)G-CSF-PEG-Fc protein complex fraction. In order to remove theimpurities, Q HP (GE healthcare, USA) anion chromatography was furtherperformed. Q HP (GE healthcare, USA) was equilibrated with a 20 mM Tris(pH 8.0) buffer solution, and then the purified ^(17,65S)G-CSF-PEG-Fcprotein complex fraction was applied thereto. Finally, a high-purity^(17,65S)G-CSF-PEG-Fc protein complex was purified with a linearconcentration gradient of a 20 mM Tris (pH 8.0) buffer solutioncontaining 1 M sodium chloride. N-terminal selectivity of the Fc regionof the prepared ^(17,65S)G-CSF-PEG-Fc protein complex was examined bypeptide mapping, and the selectivity was found to be 90% or higher.

Example 3: Preparation of Protein Complex Using PEG with DifferentReactive Groups

3-1. Preparation of ^(17,65S)G-CSF-PEG Conjugate

SMB-PEG-SMB (Nektar, USA), which is polyethylene glycol (PEG) having amolecular weight of 3.4 kDa and succinimidyl alpha-methyl butanoate(SMB) reactive groups at both ends thereof, was added to 10 mg/mL of^(17,65S)G-CSF (molecular weight 18 kDa) dissolved in 20 mM phosphatebuffer (pH 8.0) at a molar ratio of G-CSF:PEG of 1:3, and allowed toreact at room temperature under slow stifling for about 30 minutes. Toobtain a conjugate in which PEG is selectively linked to the aminoterminus of ^(17,65S)G-CSF and PEG and ^(17,65S)G-CSF are linked to eachother at a ratio of 1:1, the reaction mixture was subjected to SP HP (GEhealthcare, USA) cation exchange chromatography.

3-2. Preparation of ^(17,65S)G-CSF-PEG-Fc Complex

In order to link the ^(17,65S)G-CSF-PEG conjugate purified in Example7-1 to a region other than the N-terminus of immunoglobulin Fc, theimmunoglobulin Fc fragment was added and reacted at a molar ratio of^(17,65S)G-CSF-PEG conjugate:immunoglobulin Fc of 1:4 to 1:8. Thereaction was allowed in 20 mM phosphate buffer (pH 5.5 to 6.5) at roomtemperature for about 2 hours under slow stifling.

3-3. Isolation and Purification of ^(17,65S)G-CSF-PEG-Fc Complex

In order to remove unreacted materials and by-products after the bindingreaction of Example 7-2 and to purify the ^(17,65S)G-CSF-PEG-Fc proteincomplex thus produced, the reaction mixture was passed through a Q HP(GE Healthcare, USA) anion exchange chromatography column and thusunbound Fc was removed and a ^(17,65S)G-CSF-PEG-Fc protein complexfraction was obtained. The reaction solution was applied to a Q HPcolumn equilibrated with 20 mM Tris (pH 8.0) buffer, and the^(17,65S)G-CSF-PEG-Fc protein complex was purified with a concentrationgradient of a buffer solution containing 1 M sodium chloride (NaCl). Asmall amount of unreacted immunoglobulin Fc and ^(17,65S)G-CSF dimer asimpurities was present in the obtained ^(17,65S)G-CSF-PEG-Fc proteincomplex fraction. In order to remove the impurities, Source iso (GEHealthcare, USA) hydrophobic chromatography was further performed.Finally, a high-purity ^(17,65S)G-CSF-PEG-Fc protein complex waspurified with a linear concentration gradient of 50 mM Tris (pH 7.5)buffer solution containing 1.2 M ammonium sulfate using Source iso (GEHealthcare, USA). N-terminal selectivity of the Fc region of theprepared ^(17,65S)G-CSF-PEG-Fc protein complex was examined by peptidemapping, and the selectivity was found to be 90% or higher.

Example 4: Preparation of Protein Complex Using PEG with DifferentReactive Groups

A FacVII-ATKAVC-PEG-Fc complex was prepared using FacVII-ATKAVC, whichis a FacVII derivative of Korean Patent Application No. 10-2012-0111537previously submitted by the present inventors.

4-1. Isolation and Purification of PEG-Fc Complex

First, to link an aldehyde reactive group of maleimide-10kDa-PEG-aldehyde (NOF, Japan) to the N-terminus of immunoglobulin Fcfragment, the immunoglobulin Fc region and maleimide-10 kDa PEG-aldehydewere mixed at a molar ratio of 1:1 in a 100 mM phosphate buffer solution(pH 5.5 to 6.5), and a reducing agent, 20 mM sodium cyanoborohydride(NaCNBH3, Sigma), was added thereto under a protein concentration of 10mg/mL. The reaction was allowed at a low temperature (4° C. to 8° C.)for about 2 hours. To obtain a monoPEGylated immunoglobulin Fc fragment(maleimide-10 kDa PEG-Fc), Source Q (GE Healthcare, USA) anionchromatography was performed, and elution was performed with aconcentration gradient of sodium chloride in 20 mM Tris buffer at pH7.5.

4-2. Preparation of FacVII-ATKAVC-PEG-Fc Complex

FacVII-ATKAVC was reacted in 10 mM glycylglycine buffer at pH 5.5 atroom temperature for about 2 hours by adding 0.5 mM to 2 mMtriphenylphosphine-3,3′,3″-trisulfonic trisodium salt hydrate as areducing agent so as to reduce the C-terminus. The C-terminus-reducedFacVII-ATKAVC and monoPEGylated immunoglobulin Fc fragment (maleimide-10kDa PEG-Fc) were mixed at a molar ratio of 1:4 to 1:20, and reaction wasallowed at a total protein concentration of 1 mg/mL to 2 mg/mL in 50 mMTris buffer at pH 7.5 at room temperature for about 2 hours.

4-3. Isolation and Purification of FacVII-ATKAVC-PEG-Fc Complex

The reaction solution of Example 8-2 was subjected to Source Q anionchromatography, and the FacVII-ATKAVC-10 kDa PEG-Fc complex was elutedwith a concentration gradient of sodium chloride in a 20 mM Tris buffersolution at pH 7.5. To activate FacVII of the FacVII-ATKAVC-PEG-Fccomplex, reaction was allowed in a 0.1 M Tris-HCl buffer solution at pH8.0 under conditions of about 4 mg/mL of FacVII for about 18 hours at alow temperature (4° C. to 8° C.). Finally, high-purityFacVIIa-ATKAVC-PEG-Fc was purified by size exclusion chromatography (GEHealthcare, USA) using Superdex 200 in a 10 mM glycylglycine buffersolution at pH 5.5. N-terminal selectivity of the Fc region of theprepared FacVIIa-ATKAVC-PEG-Fe protein complex was examined by peptidemapping, and the selectivity was found to be 90% or higher.

Example 5: Preparation of Protein Complex Using PEG with a DifferentMolecular Weight

ALD-PEG-ALD (Nektar, USA), which is polyethylene glycol having amolecular weight of 10 kDa and aldehyde reactive groups at both endsthereof, was used to prepare and purify an insulin-10 kDa PEG conjugatein the same manner as in Example 5-2. The purified insulin-10 kDa PEGconjugate was concentrated to a concentration of about 5 mg/mL and thenused to prepare and purify an insulin-10 kDa PEG-Fc protein complex inthe same manner as in Example 2-3.

Example 6—Evaluation of Purity of Protein Complex

6-1. Identification of Protein Complex

The protein complexes prepared in the above Examples were analyzed bynon-reduced SDS-PAGE using a 4% to 20% gradient gel and a 12% gel.SDS-PAGE analysis and Western blot analysis of individual proteincomplexes using antibodies against immunoglobulin Fc and physiologicallyactive polypeptides were performed. As shown in FIG. 1, a couplingreaction resulted in the successful production of IFNa-PEG-Fc (A),hGH-PEG-Fc (B), ^(17,65S)G-CSF-PEG-Fc (C), Insulin-PEG-Fc (D),EPO-PEG-Fc (E), CA-Exendin4-PEG-Fc (F), and FacVII-PEG-Fc (G).

6-2. Evaluation of Purity of Protein Complex

The protein complexes prepared in the above Examples, IFNa-PEG-Fc (A),hGH-PEG-Fc (B), ^(17,65S)G-CSF-PEG-Fc (C), Insulin-PEG-Fc (D),EPO-PEG-Fc (E), and CA-Exendin4-PEG-Fc (F), were subjected to sizeexclusion chromatography, reverse phase chromatography, or ion exchangechromatography using HPLC, respectively. They displayed a single peakcorresponding to high purity of 95% or higher in each analysis.

6-3. Examination of Site Selectivity of Protein Complex

The protein complexes prepared in Examples, IFNa-PEG-Fc (A), hGH-PEG-Fc(B), ^(17,65S) G-CSF-PEG-Fc (C), insulin-PEG-Fc (D), and EPO-PEG-Fc (E),were subjected to peptide mapping analysis (reverse phasechromatography) using protease, respectively. It was confirmed that theprotein complexes linked via the N-terminus of the immunoglobulin Fcregion with high selectivity of 90% or higher were prepared.

Example 7: Comparison of Efficacy of Complex Depending on Fc BindingPosition

The protein complexes prepared in Examples, CA-Exendin4-PEG-Fc,^(17,65S)G-CSF-PEG-Fc, and EPO-PEG-Fc, were subjected to in vitro and invivo efficacy tests, respectively. As shown in the following Table,binding to the N-terminus (proline) of Fc showed better efficacy thanbinding to other regions (e.g., lysine).

TABLE 1 in vitro activity - CHO/GLP-1R bioassay of CAExendin-PEG-Fcpositional isomers % vs. EC50 Experimental Test material (ng/ml) groupCA Exendin (lysine)-PEG-(N-terminus) Fc - 95.35 100.00 Experimentalgroup CA Exendin (lysine)-PEG-(lysine) Fc 59037 16.15

As shown in Table 1, comparison of in vitro activities between CAExendin-PEG-Fc positional isomers showed that the CA Exendin-PEG-Fccomplex of the present invention, which was prepared by specific bindingto N-terminus of immunoglobulin Fc fragment, has 6 times higher potencythan a CA Exendin-PEG-Fc complex which was prepared by binding toanother position of an immunoglobulin Fc region.

TABLE 2 in vitro activity - use bone marrow cell proliferation assay of^(17,65S)G-CSF-PEG-Fc positional isomers % vs. EC50 Experimental Testmaterial (ng/ml) group ^(17,65S)G-CSF-(N-terminus)-PEG-(N- 134.43 100.00Terminus) Fc-Experimental Group ^(17,65S)G-CSF-(N-terminus)-PEG-(lysine)225.87 59.50 Fc

As shown in Table 2, comparison of in vitro activities between^(17,65S)G-CSF-(N-terminus)-PEG-(N-Terminus) Fc-Experimental GroupS-G-CSF-PEG-Fc positional isomers showed that the^(17,65S)G-CSF-(N-terminus)-PEG-(N-Terminus) Fc-Experimental GroupS-G-CSF-PEG-Fc complex of the present invention, which was prepared byspecific binding to a N-terminus of immunoglobulin Fc fragment, hasabout 67% increased titer, compared to a^(17,65S)G-CSF-(N-terminus)-PEG-(N-Terminus) Fc-Experimental GroupS-G-CSF-PEG-Fc complex which was prepared by binding to another positionof an immunoglobulin Fc region.

Meanwhile, to examine in vivo activities of the protein complex of thepresent invention, in particular, EPO-PEG-Fc positional isomers, anormocythemic mice assay was performed to measure reticulocyte levelsafter subcutaneous injection of EPO-PEG-Fc into normocythemic mice.

TABLE 3 Measurement of in vivo bio-potency reticulocyte level ofEPO-PEG-Fc positional isomers (after subcutaneous injection intononnocythemic mice). % vs. Bio-potency Experimental Test material(IU/mg) group EPO (N-terminus 84.4%)PEG- 14,189,403 100.00 (N-Terminus100%) Fc-Experimental Group EPO (N-terminus 38.2%)-PG- 225.87 59.50(lysine 83.0%) Fc

As shown in Table 3, comparison of in vivo activities between EPO-PEG-Fcpositional isomers showed that the EPO-PEG-Fc complex of the presentinvention, which was prepared by specific binding to N-terminus ofimmunoglobulin Fc fragment, has about 40% increased titer, compared toan EPO-PEG-Fc complex which was prepared by binding to another positionof an immunoglobulin Fc region.

These results suggest that when the protein complex comprising thephysiologically active polypeptide, the non-peptidyl polymer, and theimmunoglobulin Fc region is prepared by using a specific site of theimmunoglobulin Fc fragment as a binding site, the protein complex showsan improved in vivo activity of the physiologically active polypeptide.

Example 8: Randomized Human Trial ^(17,65S)G-CSF-PEG-Fc Protein Complex(EFLAPEGRASTIM) Vs. PEGFILGRASTIM in the Management ofChemotherapy-Induced Neutropenia in Breast Cancer Patients ReceivingDocetaxel and Cyclophosphamide (TC)

To evaluate the efficacy and safety of a fixed dose of EFLAPEGRASTIM(13.2 mg/0.6 mL; 3.6 mg GCSF equivalent) in patients with breast cancerwho were candidates for adjuvant or neoadjuvant chemotherapy withdocetaxel and cyclophosphamide (TC), open-label, active-controlled,human studies were conducted in 406 patients.

Eligible patients were randomized 1:1 to the following two treatmentarms: (a) EFLAPEGRASTIM Arm: EFLAPEGRASTIM 13.2 mg/0.6 mL (containing3.6 mg G-CSF) fixed dose and (b) PEGFILGRASTIM Arm: PEGFILGRASTIM 6mg/0.6 mL (containing 6.0 mg G-CSF) fixed dose. Accordingly, TC wasadministered on Day 1 of each cycle intravenously (IV) was: (i)Docetaxel at 75 mg/m² IV infusion per institute's standard of care (ii)Cyclophosphamide 600 mg/m² IV infusion per institute's standard of care.Each treatment cycle was 21 days with up to a maximum of 4 cycles ofchemotherapy. To begin full-dose chemotherapy on Day 1 of any cycle (Day22 of the previous cycle), patients must have ANC ≥1.5×10⁹/L and aplatelet count ≥100×10⁹/L.

EFLAPEGRASTIM or PEGFILGRASTIM were administered on Day 2 of each cycle,approximately 24 hours (±2 hours) after TC chemotherapy. PEGFILGRASTIMwas to be administered according to the manufacturer's PrescribingInformation (6 mg subcutaneously once per chemotherapy cycle).

Patients meeting all inclusion and exclusion criteria were randomized toeither the EFLAPEGRASTIM Arm or the PEGFILGRASTIM Arm and received studytreatment (TC) followed 24 (±2) hours by either EFLAPEGRASTIM orPEGFILGRASTIM for 4 cycles. End of treatment (EOT) visits were performed35(±5) days from the last dose of study treatment. During Cycle 1, CBCsamples were drawn on Day 1 prior to the chemotherapy and then dailyfrom Days 4 to 15 or until recovery from neutropenia. In Cycles 2 to 4,CBC samples were drawn on Day 1 predose and then on Days 4, 7, 10 and 15(±1 day for each collection). CBC was also collected at theEnd-of-Treatment Visit. Sparse PK samples for population PK werecollected in Cycle 1 on Day 2, Day 4, and Day 5 and then in Cycle 3 onDay 2, Day 4, and Day 7. Immunogenicity samples were drawn at each cyclebefore chemotherapy administration, at the end of treatment, and at 6and 12 months (long term safety).

Patients who received at least one dose of study drug and did notdiscontinue from the study are being followed for long term safety afterthe last dose of study treatment. The long-term safety includes adverseevent (AE) assessment via telephone at 3 months and 9 months and clinicvisits for AE assessment and immunogenicity blood draw at 6 months and12 months. The DSN in Cycle 1 is defined as the number of postdose daysof severe neutropenia

Efficacy analysis was conducted for the primary endpoint of the Durationof Severe neutropenia (DSN) in Cycle 1 defined as the number of postdosedays of severe neutropenia (ANC <0.5×10⁹/L) from the first occurrence ofan ANC below the threshold. The results showed that the mean DSN for theEFLAPEGRASTIM Arm was 0.20 (±0.50) days compared with a mean DSN of 0.35(±0.68) days in the PEGFILGRASTIM Arm. The difference in mean DSNbetween the EFLAPEGRASTIM Arm and the PEGFILGRASTIM Arm was −0.15 daysand the corresponding 95% CI was (−0.264, −0.032) using the percentilemethod as specified in the statistical analysis plan. Using thepre-specified criterion for the primary endpoint, the EFLAPEGRASTIM Armto the PEGFILGRASTIM Arm was demonstrated to provide non-inferior DSN(better or as effective) as PEGFILGRASTIM (upper bound of 95% CI<0.62days; p<0.0001). The results also demonstrated a statistical superiorityof EFLAPEGRASTIM over PEGFILGRASTIM in cycle 1 (upper bound of 95% CI<0;p=0.038) indicating that the incidence of severe neutropenia issignificantly lower in EFLAPEGRASTIM arm (FIG. 2 and FIG. 3). In themeantime, the incidences of adverse events were in general comparablebetween treatment groups, most of which were considered relating to thechemotherapy (TC) administration.

The Examples provided herein supports the superiority of the G-CSFprotein complex attached the immunoglobulin Fc region through a PEGmoiety to increase in vivo duration of the physiologically activepolypeptide and to increase or maintain in vivo activity (potency) atthe same time.

Based on the above description, it will be understood by those skilledin the art that the present invention may be implemented in a differentspecific form without changing the technical spirit or essentialcharacteristics thereof. Therefore, it should be understood that theabove embodiment is not limitative, but illustrative in all aspects. Thescope of the invention is defined by the appended claims rather than bythe description preceding them, and therefore all changes andmodifications that fall within metes and bounds of the claims, orequivalents of such metes and bounds, are therefore intended to beembraced by the claims.

What is claimed is:
 1. A method for treating or preventing neutropeniain a patient receiving chemotherapy, comprising administering to thepatient a therapeutically effective amount of a protein complexcomprising a modified human granulocyte-colony stimulating factor(hG-CSF) covalently linked to an N-terminus of an immunoglobulin Fcregion via a non-peptidyl polymer, wherein the modified hG-CSF comprisesthe amino acid sequence of SEQ ID NO:
 1. 2. The method of claim 1,wherein the protein complex is administered to the patient within about6 hours of completion of the chemotherapy.
 3. The method of claim 1,wherein the protein complex is administered to the patient within about5 hours of completion of the chemotherapy.
 4. The method of claim 1,wherein the protein complex is administered to the patient within about2 hours of completion of the chemotherapy.
 5. The method of claim 1,wherein the protein complex is administered to the patient within about1 hour of completion of the chemotherapy.
 6. The method of claim 1,wherein the neutropenia is severe chronic neutropenia or febrileneutropenia.
 7. The method of claim 1, wherein the chemotherapy isadjuvant or neoadjuvant chemotherapy.
 8. The method of claim 1, whereinthe adjuvant or neoadjuvant chemotherapy is a combination of docetaxeland cyclophosphamide.
 9. The method of claim 1, wherein thetherapeutically effective amount is a unit dosage form selected from 25μg/kg, 50 μg/kg, 100 μg/kg, and 200 μg/kg.
 10. The method of claim 1,wherein both ends of the non-peptidyl polymer are respectively linked tothe modified hG-CSF and the immunoglobulin Fc region through reactivegroups by a covalent bond.
 11. The method of claim 1, wherein theimmunoglobulin Fc region comprises an immunoglobulin.
 12. The method ofclaim 1, wherein the immunoglobulin Fc region is aglycosylated.
 13. Themethod of claim 1, wherein the immunoglobulin Fc region comprises one ormore of CH2, CH3, and CH4 domains.
 14. The method of claim 1, whereinthe immunoglobulin Fc region comprises CH2 and CH3 domains.
 15. Themethod of claim 1, wherein the immunoglobulin Fc region furthercomprises a hinge region.
 16. The method of claim 1, wherein each domainof the immunoglobulin Fc fragment is a hybrid of domains, each of thedomains having a different origin derived from immunoglobulins selectedfrom IgG, IgA, IgD, IgE, and IgM.
 17. The method of claim 1, wherein theimmunoglobulin Fc fragment comprises an IgG4 Fc fragment.
 18. The methodof claim 1, wherein the non-peptidyl polymer is selected frompolyethylene glycol, polypropylene glycol, an ethylene glycol-propyleneglycol copolymer, polyoxyethylated polyol, polyvinyl alcohol,polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer,a lipid polymer, chitin, hyaluronic acid, and a combination thereof. 19.The method of claim 1, wherein the non-peptidyl polymer comprisespolyethylene glycol.
 20. The method of claim 19, wherein thepolyethylene glycol has a molecular weight of 3.4 kDa.